The current studies are a multiple-P.I. proposal that focuses on the role of mTOR in oligodendrocyte development and CNS myelination. This is an important research area because of the devastating consequences of demyelination in humans and the need to understand the molecular details of how myelination and remyelination are regulated, in order to repair such damage. The Macklin laboratory has investigated the role of Akt and mTOR in CNS myelination, while the Wood laboratory has investigated the role of mTOR in oligodendrocyte differentiation and has identified an mTOR-regulated proteome in oligodendrocytes. There is inconsistency in the literature and in the preliminary data from the two laboratories as to when in the developmental program of oligodendrocytes mTOR becomes a major regulator. Thus, the current proposal is designed to answer unequivocally when and how the mTOR signaling complexes regulate oligodendrocyte development including potential actions on both differentiation and myelination. Rather than compete to address these questions, we propose a collaborative project using complementary mouse lines, and standardized reagents and techniques. The two laboratories have complementary sets of conditional mutant mice that will be used collaboratively to investigate these questions. In the first specific aim, we will investigate the mechanisms by which mTOR regulates oligodendrocyte differentiation, testing the hypothesis that mTOR directly regulates oligodendrocyte differentiation via specific actions of both mTORC1 and mTORC2. This will be investigated by studying the signaling pathways and the differentiation events that are modulated in mTOR, raptor or rictor conditionally-deleted mice. In the second specific aim, we will investigate the mechanisms by which mTOR regulates CNS myelination and myelin maintenance. We will test the hypothesis that mTOR directly regulates myelination via both mTORC1 and mTORC2, with differential control by each complex. Studies will additionally investigate how active myelination shifts to myelin maintenance in the CNS. In the third specific aim, we will investigate the upstream regulation of the two mTOR complexes by TSC1/2 in developing oligodendrocytes. These studies will test the hypothesis that TSC signaling regulates oligodendrocyte differentiation and CNS myelination through upstream inhibition of mTORC1 and activation of mTORC2. TSC1/2 are considered to be negative regulators of mTOR signaling, yet in some contexts loss of TSC activity induces hypomyelination rather than the expected hypermyelination. Establishing how they impact mTOR signaling in the oligodendrocyte is therefore important. In the final aim, we will determine the mechanisms by which the mTOR pathway regulates remyelination. The crucial questions in this aim will be whether the role of this pathway in the regulation of oligodendrocyte differentiation and myelination recapitulates its function during development, or whether there are unique elements of mTOR regulation of remyelination in adult tissue. This aim clearly has significant impact on our understanding of remyelination in multiple sclerosis. PUBLIC HEALTH RELEVANCE: Myelination is essential for normal nervous system development, and demyelination in the adult causes serious diseases such as multiple sclerosis. The current proposed studies address the intracellular signaling pathways that regulate central nervous system myelination during oligodendrocyte development, and following a demyelinating event in the adult.